SUMMARY There is a growing interest to understand how painful events in the neonatal period affect the development of nociceptive neural circuitry and alters pain perception in adulthood. Accumulating evidence from clinical studies indicates that the pain experience as a child has a significant impact on the pain responses at adulthood. Pre- clinical studies also clearly demonstrated that the exposure to noxious stimuli early in life causes long-term alterations in sensory processing. However, the etiology of chronic pelvic pain (CPP) is complex and poorly defined. The NIDDK has estimated that CPP is responsible for 4,137,000 outpatient or clinic visits/year and about 90% of them are female. Recent study also indicates that estimated medical cost for treating CPP exceeds $2 billion/year. The focus of our ongoing NIH application (R01 DK099201-01A1) is to study how the pain signaling pathway from the bladder to the spinal cord alters following neonatal bladder inflammation in rats. During this funding period, we have established for the first time the involvement of miRNAs (noncoding small RNAs) in post-transcriptional suppression of the developing spinal GABAergic system and long-term visceral hypersensitivity in a model of neonatal zymosan-induced cystitis. However, little is known regarding the involvement of supraspinal descending pain modulatory systems in spinal hyperexcitability following this early- life induced cystitis in rats. In this competitive renewal application, we would like to extend our study to investigate further the contribution of higher brain regions including midbrain PAG-RVM axis in the development of CPP in neonatal-cystitis rats. We will test the hypothesis that the long-lasting spinal sensitization following intense painful visceral stimulus in early-life is due to i) altered functional connectivity between PAG and higher brain regions ii) altered functional characteristics of RVM neurons projecting to spinal cord, and (iii) miRNA-mediated post-transcriptional dysregulation of pain modulatory neurons in the RVM. The proposed experiments will be systematic investigation to explore the intrinsic neuromolecular mechanisms and functional changes of descending pain modulation in neonatal cystitis-induced CPP. The identification of differentially expressed RVM miRNAs and molecular characterization of neuronal plasticity in the RVM neuron are of significance in designing receptor and/or miRNA-based pharmacological manipulation for better detection and therapeutic targeting of CPP.